A new lipase (Alip2) with high potential for enzymatic hydrolysis of the diester diethyladipate to the monoester monoethyladipate
Introduction
Monoesters of symmetrical dicarboxylic acids such as monoethyl adipate (MEA) of diethyl adipate (DEA) can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications and there is an increased demand for those substances.
Polymers with functional groups, known as functional polymers, are of increasing interest in different areas including sensors, energy storage, food, biomedical and environmental applications [1]. Spacer groups are often an essential part of these polymers. They serve as ‘distance holder’ between polymer material and the functional group to ensure the accessibility and improve the binding ability. The length and type of the respective spacer arm determines chemical, physical and biological properties as well as the flexibility and the formability [2].
In addition, dicarboxylic acid monoesters and diol monoesters are key substances in the pharmaceutical industry or serve as building blocks in the chemical industry [[3], [4], [5]].
Monoesters can be synthesized chemically by (1) acid or base catalyzed hydrolysis of the diesters, (2) esterification of a dicarboxylic acid with an alcohol or diols with carboxylic acids and (3) transesterification of the diester. But these reactions are often not selective: both ester bonds can be cleaved or both carboxyl or hydroxy groups can be (trans)esterified. This is leading to a mixture of the monoester, diester and diacid and thus monoester yields are low depending on the method used. The educt’s structure also affects the yield and selectivity of the reaction products, as it was shown for the microwave assisted hydrolysis of carboxylic esters by InI3/SiO2/H2O yielding between 70 and 93 % depending of the length and structure of the substrate [6].
In contrast to chemical synthesis, enzymatic reactions have many advantages. They can be selective, so that high monoester concentrations can be reached. The affinity and rate for the diester should be much higher than for the monoester to obtain especially monoester as a product.
Enzymatic syntheses for the preparation of dicarboxylic acid monoesters and diol monoesters have already been used successfully with yields up to 100 % [[4], [5], [6], [7], [8]]. In particular, lipases (EC 3.1.1.3) are currently used as biocatalysts for the efficient production of dicarboxylic acid monoesters by the selective hydrolysis of diesters [4,5,[7], [8], [9], [10]].
The yeast Blastobotrys raffinosifermentans (syn. Arxula adeninivorans) LS3 [11] offers a high potential to uncover of new lipases because the yeast can use, among other substrates, a number of lipophilic compounds as the sole source of energy and carbon [12]. The thermo- and halotolerant yeast was already used in the 1980s for ‘single cell protein production’ in Malchin (Mecklenburg-West Pomerania, Germany) because of its excellent growth parameters. In addition, its C and N source spectrums are exceptionally extensive for yeast. Based on these properties, B. raffinosifermentans has been used as a producer of a number of proteins [13], in particular such as Interleukin-6 [14] and Human Serum Albumin [[15], [16], [17]], and enzymes such as the feed additive phosphatase APHO1 with phytase activity [18], tannase [19,20] and the extracellular lipase Alip1p [12].
In this study, the genome of B. raffinosifermentans LS3 was screened for putative lipases with the ability for monoester production. After a selection, the Blastobotrys-endogenous, putative lipase Alip2p was purified, characterized and its potential for the production of the monoester monoethyl adipate (MEA) of the symmetrical dicarboxylic acid diethyl adipate (DEA) by hydrolysis was evaluated.
Section snippets
Chemicals
DES, DAH, DMSub, DMA, DEA, DEM, Sebacic acid, MEA, Ethylhydrosuccinate were purchased from Alfa Aesar. DAB, DESeb, Monomethyladipate, DIpA, DMSeb, DAD, Adipic acid, malonic acid, succinic acid and suberic acid was bought by TCI Chemicals (abbreviations see Table 5). Diols 1,6-hexandiol, 1,4-butandiol and 110-decandiol were from Acros.
Strain and culture conditions
Escherichia coli strain XL1 blue [recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac [F’proABlacl q Z DM15 Tn10 (Tetr)], from Invitrogen (Grand Island, NY,
Identification and analysis of the ALIP2 gene of B. raffinosifermentans
In the genome of B. raffinosifermentans LS3 several putative lipase genes were annotated by using a computed set of concatenated orthologs and a super tree approach combining all individual gene phylogenies [35]. The corresponding genes of all identified putative lipases were overexpressed in the yeast itself and crude extracts or culture supernatant for the enzymes with secretion signal were screened for pNP-butyrate and DEA hydrolysis (data not shown). Highest activities and MEA yields were
Discussion
There exists a bulk of known lipases used in a great variety of different applications [[36], [37], [38]]. Some lipases even selectively hydrolyze symmetric diesters into monoesters [39,40]. The non-conventional yeast B. raffinosifermentans LS3 produces many lipases, too. In this study, the B. raffinosifermentans lipase coding gene sequences were identified, isolated, produced in the yeast itself and screened for the hydrolysis of aliphatic diesters, especially DEA.
The identified putative
Conclusions
The intracellular lipase Alip2p from the yeast B. raffinosifermentans represents a promising enzyme candidate for the efficient monohydrolysis of the symmetrical DEA into MEA as well as further dicarboxylic esters and diol diesters into the corresponding monoesters. A yield of 96 % for MEA in only 30 min could be achieved with less than 2 % of the diacid AA detectable. During this period, a higher yield of MEA could also not be obtained with any commercial lipase tested. The use of lipase
Author contributions
Daniela Nietz: Conceptualization, Methodology, Investigation, Validation, Visualization, Writing.
Rüdiger Bode: Design of the study, Validation, biochemical characterisation.
Gotthard Kunze: Supervision, Project administration, Funding acquisition.
Marion Rauter: Conceptualization, Methodology, Investigation, Designed and performed experiments.
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgments
We are grateful to Dr. Hans-Matthias Vorbrodt and Dr. Karin Becker for the helpful discussions and Dr. Matthew Haas for critical reading of the manuscript. We also thank Carla Liebig and David Sjaba for excellent technical assistance. The research work was supported by a grant (grant no. KF2131630CS) from BMWi.
References (57)
- et al.
Two enzymatic procedures for the selective synthesis of malic acid monoesters
Enzyme Microb. Technol.
(1999) - et al.
Enzymatic synthesis of lactate and glycolate esters of fatty alcohols
Enzyme Microb. Technol.
(1999) - et al.
Enantioselective desymmetrization of prochiral diesters catalyzed by immobilized Rhizopus oryzae lipase
Tetrahedron Asymmetry
(2011) - et al.
Asymmetric desymmetrization of dialkyl bicyclo[2.2.1]hept-2,5-diene-2,3-dicarboxylates by a thermophilic esterase/lipase
Tetrahedron Asymmetry
(2002) - et al.
Regio- and enantioselectivity of the enzyme catalysed hydrolysis of citric acid derivatives
Tetrahedron Asymmetry
(1998) - et al.
High-level production and secretion of recombinant proteins by the dimorphic yeast
FEMS Yeast Res.
(2002) - et al.
The ALEU2 gene - A new component for an Arxula adeninivorans-based expression platform
FEMS Yeast Res.
(2003) - et al.
Synthesis of benzyl β-d-galactopyranoside by transgalactosylation using a β-galactosidase produced by the over expression of the Kluyveromyces lactis LAC4 gene in Arxula adeninivorans
J. Mol. Catal., B Enzym.
(2013) - et al.
Studies on transformation of Escherichia coli with plasmids
Jmb
(1983) - et al.
Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae - Post-translational modifications are dependent on the host strain
Biochim. Biophys. Acta - Gene Struct. Expr.
(1998)
A rapid and sensitive method for the quantiation of microgram quantities of protein utilizing the principle of dye-binding
Anal. Biochem.
A buffer solution for colorimetric comparison
J. Biol. Chem.
Lipase catalysis in organic solvents: advantages and applications
Biol. Proced. Online
Lipases for biotechnology
Curr. Opin. Biotechnol.
Lipolytic activity of Williopsis californica and Saccharomyces cerevisiae in extra virgin olive oil
Int. J. Food Microbiol.
Screening of yeast and fungal strains for lipolytic potential and determination of some biochemical properties of microbial lipases
J. Mol. Catal. - B Enzym.
Bacterial lipases
Screening of microbes for lipases specific for saturated medium and long-chain fatty acids of milk fat
Int. Dairy J.
Aroma synthesis by immobilized lipase from Mucor sp
Enzyme Microb. Technol.
Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125
J. Mol. Catal. B Enzym.
Engineering redox balance through cofactor systems
Trends Biotechnol.
Quantification of metabolic limitations during recombinant protein production in Escherichia coli
J. Biotechnol.
ADH from Rhodococcus ruber expressed in Arxula adeninivorans for the synthesis of 1- (S) -phenylethanol
J. Mol. Catal. B, Enzym.
Smart functional polymers - A new route towards creating a sustainable environment
RSC Adv.
Spacer groups in macromolecular structures
Polimeri
Enzyme-Catalyzed Hydrolysis of Bicycloheptane and Cyclobutene Diesters to Monoesters
Org. Process Res. Dev.
Enzymatic desymmetrization of dimethyl Cylcohex-4-ene- cis -1,2-dicarboxylate to (1 S, 2 r)-2-(methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid
Org. Process Res. Dev.
An efficient and general method for Ester Hydrolysis on the surface of silica gel catalyzed by indium triiodide under microwave irradiation
Synth. Commun. An Int. J. Rapid Commun. Synth. Org. Chem.
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2023, International Journal of Biological MacromoleculesRate equations for two enzyme-catalyzed Ping Pong bi bi reactions in series: General formulation for two reaction loops joined by a common vertex and deduction of a reaction loop selectivity factor
2022, Biochemical Engineering JournalCitation Excerpt :One example involving hydrolysis is the hydrolysis of biphenyl esters to produce asymmetric building blocks that have a wide range of applications in chemical synthesis [4]. Another example is the hydrolysis of diethyl adipate, to produce monomeric spacer groups for functional polymers [5]. The reactions shown in Fig. 1 involve two sequential reaction loops, each involving catalysis by the Ping Pong bi bi mechanism.